A dimeric proteomimetic prevents SARS-CoV-2 infection by dimerizing the spike protein.

Protein tertiary structure mimetics are valuable tools to target large protein-protein interaction interfaces. Here, we demonstrate a strategy for designing dimeric helix-hairpin motifs from a previously reported three-helix-bundle miniprotein that targets the receptor-binding domain (RBD) of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Through truncation of the third helix and optimization of the interhelical loop residues of the miniprotein, we developed a thermostable dimeric helix-hairpin. The dimeric four-helix bundle competes with the human angiotensin-converting enzyme 2 (ACE2) in binding to RBD with 2:2 stoichiometry. Cryogenic-electron microscopy revealed the formation of dimeric spike ectodomain trimer by the four-helix bundle, where all the three RBDs from either spike protein are attached head-to-head in an open conformation, revealing a novel mechanism for virus neutralization. The proteomimetic protects hamsters from high dose viral challenge with replicative SARS-CoV-2 viruses, demonstrating the promise of this class of peptides that inhibit protein-protein interaction through target dimerization.

Moonlighting Biochemistry of Cysteine Synthase: A Species-specific Global Regulator.

Cysteine Synthase (CS), the enzyme that synthesizes cysteine, performs non-canonical regulatory roles by binding and modulating functions of disparate proteins. Beyond its role in catalysis and regulation in the cysteine biosynthesis pathway, it exerts its moonlighting effect by binding to few other proteins which possess a C-terminal "CS-binding motif", ending with a terminal ILE. Therefore, we hypothesized that CS might regulate many other disparate proteins with the "CS-binding motif". In this study, we developed an iterative sequence matching method for mapping moonlighting biochemistry of CS and validated our prediction by analytical and structural approaches. Using a minimal protein-peptide interaction system, we show that five previously unknown CS-binder proteins that participate in diverse metabolic processes interact with CS in a species-specific manner. Furthermore, results show that signatures of protein-protein interactions, including thermodynamic, competitive-inhibition, and structural features, highly match the known CS-Binder, serine acetyltransferase (SAT). Together, the results presented in this study allow us to map the extreme multifunctional space (EMS) of CS and reveal the biochemistry of moonlighting space, a subset of EMS. We believe that the integrated computational and experimental workflow developed here could be further modified and extended to study protein-specific moonlighting properties of multifunctional proteins.

Desolvation of Peptide Bond by O to S Substitution Impacts Protein Stability.

Amino acid side chains are key to fine-tuning the microenvironment polarity in proteins composed of polar amide bonds. Here, we report that substituting an oxygen atom of the backbone amide bond with sulfur atom desolvates the thioamide bond, thereby increasing its lipophilicity. The impact of such local desolvation by O to S substitution in proteins was tested by synthesizing thioamidated variants of Pin1 WW domain. We observe that a thioamide acts in synergy with nonpolar amino acid side chains to reduce the microenvironment polarity and increase protein stability by more than 14 °C. Through favorable van der Waals and hydrogen bonding interactions, this single atom substitution significantly stabilizes proteins without altering the amino acid sequence and structure of the native protein.

Molecular mechanism of selective substrate engagement and inhibitor disengagement of cysteine synthase.

O-acetyl serine sulfhydrylase (OASS), referred to as cysteine synthase (CS), synthesizes cysteine from O-acetyl serine (OAS) and sulfur in bacteria and plants. The inherent challenge for CS is to overcome 4 to 6 log-folds stronger affinity for its natural inhibitor, serine acetyltransferase (SAT), as compared with its affinity for substrate, OAS. Our recent study showed that CS employs a novel competitive-allosteric mechanism to selectively recruit its substrate in the presence of natural inhibitor. In this study, we trace the molecular features that control selective substrate recruitment. To generalize our findings, we used CS from three different bacteria (Haemophilus, Salmonella, and Mycobacterium) as our model systems and analyzed structural and substrate-binding features of wild-type CS and its ∼13 mutants. Results show that CS uses a noncatalytic residue, M120, located 20 Šaway from the reaction center, to discriminate in favor of substrate. M120A and background mutants display significantly reduced substrate binding, catalytic efficiency, and inhibitor binding. Results shows that M120 favors the substrate binding by selectively enhancing the affinity for the substrate and disengaging the inhibitor by 20 to 286 and 5- to 3-folds, respectively. Together, M120 confers a net discriminative force in favor of substrate by 100- to 858-folds.